The objective of the proposed research is to determine the pathways of biosynthesis of both the pyrimidine and thiazole moieties of thiamine, vitamin B1. The pyrimidine moiety 2-methyl-4-amino-5-hydroxymethylpyrimidine (MAHMP) is known to be synthesized from an intermediate of purine biosynthesis, aminoimidazole ribotide (AIR), but the steps between AIR and MAHMP are unknown. The thiazole moiety, 4-methyl-5-beta-hydroxyethylthiazole (MHET), has a totally unknown biosynthetic pathway. The problem will be approached using Salmonella typhimurium or Escherichia coli mutant strains which will accumulate either MAHMP, MHET or the intermediates in their pathways. Initially attempts will be made to obtain mutants which produce excessive quantities of thiamine or its precursors. Other mutants to be used will have blocks in the MHET pathway and between AIR and MAHMP. Additionally, mutants which are also blocked in the conversion of AIR to purines (called purE mutants) will be employed. These will produce increased amounts of MAHMP and its precursors since the competing reaction for AIR is eliminated. The mutants will be used in studies to determine the origins of the 2-methyl group, carbon-5 and the hydroxymethyl carbon of MAHMP. These experiments will involve the use of 13C- or 14C-labelled precursors, and the MAHMP produced will be analyzed by carbon magnetic resonance or by radioactive counting techniqes. Mutants blocked between AIR and MAHMP will be used with (14C) AIR to accumulate and identify intermediates in this pathway. Possible intermediates will be chemically synthesized and fed to these mutants to determine if any of the compounds are converted to MAHMP. Those mutants blocked in the formation of MHET will be used to accumulate and identify intermediates in that pathway again by making use of carbon-14. The possible role of hydroxybenzothiazole as an MHET precursor will be investigated by determining if hydroxybenzothiazole can satisfy the MHET requirement and by determining if it can be converted to MHET.